Isolation of Adhirons Specific for Plant Protoplast Membrane Biomarkers Is Simplified by Phagemid Design.

Phage display is an effective method to retrieve binders specific for a target epitope from a large clone library. Nevertheless, the panning process allows for the accumulation of some contaminant clones into the selected phage pool and, consequently, each clone requires individual screening to verify its actual specificity. This step is time-consuming, independently on the chosen method, and relies on the availability of reliable reagents. Since phages display a single binder responsible for the antigen recognition but their coat is formed by several repeats of the same proteins, the targeting of coat epitopes is often exploited to amplify the signal. Commercial anti-M13 antibodies are commonly labeled with peroxidase or FITC but customized antibodies might be necessary for specific applications. Here, we report a protocol describing the selection of anti-protoplast Adhirons that relies on the availability of nanobodies fused to a fluorescent protein to use during flow cytometry screening. Specifically, when preparing our Adhiron synthetic library, we designed a new phagemid that allows the expression of the clones fused to three tags. These can interact with a large variety of commercial and home-made reagents, selected according to the needs of the downstream characterization process. In the described case, we combined the ALFA-tagged Adhirons with an anti-ALFAtag nanobody fused with the fluorescent protein mRuby3.